Our long-term goal is to study mechanisms mediating hyperglycemia caused nephropathy in diabetes mellitus. This proposal will test a hypothesis that eNOS (endothelial nitric oxide synthase) of glomerular endothelia is a direct target of hyperglycemia. Our preliminary results suggest that high glucose inhibits glomerular NO production, and the eNOS may be the only NOS expressed in glomeruli of normal kidney. We ask whether the glomerular eNOS is a direct target of hyperglycemia, what possible mechanisms mediate this process, and what are consequences of eNOS inhibition on glomerular function. Three methods have been developed for the project: direct measurement of glomerular NO production with NO-sensitive fluorescent dye (DAF2-DA) and confocal microscopy, NO-sensitive microelectrode measurement on kidney slices, and an in vivo confocal microscopy imaging of mouse kidney to evaluate glomerular permeability in real time. AIM-1 asks if high glucose can acutely affect glomerular NO production in normal kidney, and if this effect is mediated by PKC activation. Experiments will test effect of high glucose (20mM-30mM) on glomerular NO production in kidney slices of normal ICR mice. Role of PKC-beta activation in mediating the high glucose effect on the NO production will be tested using specific PKC inhibitors. The possibility of high glucose altered oxidative activity in glomeruli will also be tested. AIM-2 asks whether hyperglycemia affects glomerular permeability in vivo. Experiments will determine whether change glomerular NO production can affect glomerular permeability; whether hyperglycemia increases glomerular permeability through inhibition of glomerular eNOS, and whether PKC activation mediating the hyperglycemia-induced glomerular functional change examined by in vivo confocal imaging. These experiments on normal mice with experimentally induced hyperglycemia will help us understanding possible mechanisms of hyperglycemia inhibiting glomerular eNOS, and its contribution to initiate glomerular complications in diabetes mellitus. [unreadable] [unreadable]